Eukaryotic initiation factor 2 from rat liver: no apparent function for the β-subunit in the formation of initiation complexes

Eukaryotic protein synthesis initiation factor 2 (eIF-2) from rat liver has been resolved into two subfractions by anion-exchange chromatography on DEAE-cellulose. One of these contained all three components (eIF-2α, eIF-2β, eIF-2γ) characteristic of mammalian eIF-2, whilst the other fraction contained only two. By a number of criteria these were shown to be eIF-2α and eIF-2γ. The absence of eIF-2β from this fraction was not due to its proteolytic degradation during purification since it was unaffected by the inclusion of a range of proteinase inhibitors in the isolation media. The properties of eIF-2 containing or lacking eIF-2β have been directly compared. It was found that eIF-2β was not required for the binding of guanine nucleotides to eIF-2 or for formation of ternary initiation complexes with GTP and the initiator tRNA. eIF-2 lacking eIF-2β was able to form 40 S initiation complexes and the presence of eIF-2β was also unnecessary for the stimulation of eIF-2 activity by the recycling factor, eIF-2B. Some of these findings are at variance with previous reports in which eIF-2β was removed proteolytically. The role of eIF-2β in the overall physiological function of eIF-2 remains to be elucidated.     

Identification and quantitation of levels of protein synthesis initiation factors in crude HeLa cell lysates by two-dimensional polyacrylamide gel electrophoresis

Protein synthesis initiation factors in purified preparations and in crude lysates of HeLa cells were fractionated by two-dimensional polyacrylamide gel electrophoresis in order to characterize their molecular forms. Specific spots in the complex cytoplasmic protein gel pattern which corresponded to the initiation factor proteins were identified by co-migration of purified initiation factors with 35S-labeled cell lysates, partial proteolytic digestion mapping, and immunoblotting analysis using antisera or affinity-purified antibodies to the initiation factors. Spots identified as eukaryotic initiation factor (eIF) 2 alpha, eIF-2 beta, eIF-2 gamma, eIF-4A, and four eIF-3 proteins of less than 50,000 Da corresponded to moderately abundant lysate proteins. Minor isoelectric variant forms of eIF-2 beta, eIF-2 gamma, and eIF-4A were detected by immunoblot analysis of lysate proteins, suggesting either covalent modification of these factor proteins or contaminating antibodies. eIF-2 beta and eIF-4B were present in at least two isoelectric forms, confirming covalent modification of these proteins. The cellular levels of the initiation factor proteins were measured by excising and counting radioactivity in gel-resolved spots corresponding to factors in lysates labeled in vivo. The individual factor protein abundancies span nearly a 10-fold range, from 1.1 to 9.8 million molecules/cell. The factor to ribosome ratio for eIF-2 was 0.8, for the average eIF-3 protein about 0.6, and for eIF-4A it was significantly higher at 3.0.     

Shape and location of eukaryotic initiation factor eIF-2 on the 40S ribosomal subunit of rat liver. Immunoelectron-microscopic and hydrodynamic investigations

The location of initiation factor eIF-2 and of its subunits in quaternary initiation complexes (40S-ribosomal-subunit.eIF-2. GuoPP[CH2]P.Met-tRNAf) was investigated by immunoelectron microscopy. Quaternary complexes were fixed with glutaraldehyde and reacted with affinity-purified polyclonal antibodies against eIF-2 alpha, eIF-2 beta or eIF-2 gamma. The dimeric immune complexes obtained by sucrose gradient centrifugation were investigated electron microscopically after negative staining. Antibody-binding sites were observed on the interface side of the 40S ribosomal subunit in the region between the 'head' and the 'body' (neck region) of the 40S ribosomal subunit. Within this region, eIF-2 alpha points to the rear side, whereas eIF-2 beta and eIF-2 gamma point to the frontal side of the 40S subunit indicating an elongated shape of eIF-2 about 15 nm long. By analytical ultracentrifugation of isolated eIF-2 the sedimentation and diffusion coefficients were determined to be 6.54 S and 4.74 x 10(-7) cm2/s respectively. From these data, a molar mass of 122.4 kg/mol and a dry volume of 147.4 nm3 were calculated. For the shape of eIF-2 a prolate ellipsoid of revolution is assumed with a maximal length of about 15 nm and with an axial ratio of about 1:3.5. This conclusion is further confirmed by a calculated frictional ratio of 1.37 and a Stokes radius of about 4.54 nm.     

Purification and characterization of eukaryotic initiation factor 2 and a guanine nucleotide exchange factor from rat liver

Two polypeptide chain initiation factors, eukaryotic initiation factor 2 (eIF-2) and guanine nucleotide exchange factor (GEF), were isolated from rat liver. Two forms of eIF-2 were identified, one contained three subunits (alpha, beta, and gamma), and the other contained only the alpha- and gamma-subunits. The three-subunit form was similar to eIF-2 from rabbit reticulocytes with respect to the sedimentation coefficient, Stokes radius, molecular weight of the alpha- and gamma-subunits, ability to restore protein synthesis in hemin-deficient reticulocyte lysate, and immunological cross-reactivity of the alpha-subunits using antibodies against liver eIF-2. In contrast, the beta-subunits of the liver and reticulocyte factors were distinct; they had different molecular weights, and antibodies against rat liver eIF-2 beta did not recognize the beta-subunit of the reticulocyte factor. Furthermore, the GDP dissociation constant for reticulocyte eIF-2 was more than twice that of the liver factor. GEF from rat liver reversed GDP inhibition of the ternary complex assay and catalyzed the exchange of eIF-2-bound GDP for free GDP or GTP, characteristics ascribed to the corresponding protein from rabbit reticulocytes. However, its subunit composition and molecular weight were different from those reported for reticulocyte GEF. The T1/2 for GDP exchange mediated by GEF was about 5-fold slower with two-subunit than with three-subunit eIF-2. In addition, the KD for GDP was lower for two-subunit than for three-subunit eIF-2 when GEF was present. Taken together, these data demonstrate species-associated variability in the beta-subunit of eIF-2 and suggest a crucial role for the beta-subunit in the functional interaction of eIF-2 and GEF.     

Regulation of initiation factors during translational repression caused by serum depletion. Covalent modification

One to 2 h after transfer of HeLa cells into fresh serum-containing medium, when translation rates are maximal, the initiation factor proteins were examined on immunoblots of two-dimensional gels. Eukaryotic initiation factor (eIF)-2 alpha, eIF-2 beta, and eIF-4A each formed a single immunoreactive spot; eIF-2 gamma formed 2 spots; and eIF-4B formed a complex array of 12-20 spots. After 4 days of growth in unreplenished medium, when translation rates have dropped 4-6-fold, several alterations in the isoelectric forms were observed: eIF-2 alpha now occurred in 2 forms, eIF-2 beta was present in 3-4 forms, and the most acidic cluster of eIF-4B variants was decreased or absent while a new isoelectric variant appeared at the basic end of the array. No changes were observed for eIF-2 gamma or eIF-4A. The 35-50-kDa subunits of the multiprotein initiation factor eIF-3 also showed no changes when the aforementioned growth states were compared. Resolution of 32P-labeled lysates by isoelectric focusing/sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that the eIF-2 alpha modification and the loss of eIF-4B variants reflected changes in phosphorylation states. Stimulation of 4-day grown cells with fresh serum-containing medium caused a reversal of the initiation factor modifications back to the forms prevailing shortly after replating. This analysis indicates that covalent modifications appear concurrently with decreasing initiation rates and suggests that they may be causative.     

The purification and characterization of subunits alpha, beta, and gamma from the rabbit reticulocyte eukaryotic initiation factor 2

Eukaryotic initiation factor 2 (eIF-2) contains three nonidentical subunits, alpha, beta, and gamma. The simultaneous purification of all three subunits was achieved by reverse-phase HPLC using a 0.1% trifluoroacetic acid-acetonitrile binary solvent system. The order of the eluted subunits, beta, alpha, and gamma, was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. After hydrolysis in 6 N HCl, picomole level amino acid composition analysis was achieved by the ninhydrin reaction on a Beckman 6300 system. Using second-derivative spectroscopic analysis, Trp was detected in all three subunits. All three subunits were subjected to amino-terminal sequence analysis. The amino-terminal of eIF-2 alpha from amino acid positions 1 to 23 inclusive was determined. The order of eight amino acids from the amino-terminal of eIF-2 gamma was also determined. This characterization and partial determination of the primary sequence of these subunits permit the utilization of molecular biology techniques in order to elucidate the complete primary structure. Additionally, the partial amino acid sequence data permitted the designation of synthetic gene probes as well as the identification of eIF-2 alpha and gamma cDNA and/or genomic clones.     

Chemical modification of pig liver initiation factor eIF-2 with N-ethylmaleimide. Amino acid sequences around the N-ethylmaleimide-reactive sulfhydryl groups and the effect of GDP on the modification

The activity of eukaryotic initiation factor eIF-2 as to the formation of the ternary complex, eIF-2 GTP Met-tRNA(f), is inhibited by N-ethylmaleimide. Our preparation of pig liver eIF-2 contained alpha and gamma subunits and was inhibited by more than 90% by N-ethylmaleimide. Using our eIF-2, we determined the sequences around the N-ethylmaleimide-reactive sulfhydryl groups, studied the effect of GDP on the sulfhydryl modification and that of NEM on the [3H]GDP binding, and examined the protective effect of GTP against the inhibition of ternary complex formation by N-ethylmaleimide. Both subunits of native eIF-2 contained [14C]N-ethylmaleimide-reactive sulfhydryl groups. One N-ethylmaleimide-reactive sulfhydryl group was in the alpha subunit and 4 were in the gamma subunit. The sequence of the peptide of the alpha subunit was determined to be: Ala-Gly-Leu-Asn-Cys-Ser-Thr-Glu-Thr-Met-Pro-Ile. Two of the four [14C]N-ethylmaleimide-reactive sulfhydryl groups in the gamma subunit were highly reactive, their sequences being: Ile-Val-Leu-Thr-Asn-Pro-Val-Cys-Thr-Glu-Val-Gly-Glu-Lys (gamma 1); Ser-Cys-Gly-Ser-Ser-Thr-Pro-Asp-Glu-Phe-Pro-Thr-Asp-Ile-Pro-Gly-Thr-Lys (gamma 3a). Peptide gamma 3a contained the consensus sequence element (AspXaaXaaGly) of GTP-binding proteins. With preincubation of eIF-2 with GDP, the incorporation of [14C]N-ethylmaleimide into the gamma subunit was reduced to 40% of the control level, but the 14C-incorporation into the alpha subunit did not change.(ABSTRACT TRUNCATED AT 250 WORDS)     

Interaction of wheat germ translation initiation factor 2 with GDP and GTP.

The wheat germ translation initiation factor 2 (WGeIF-2) was isolated in a homogeneous state by an efficient procedure and characterized. Its molecular mass, as determined by a gel-filtration method is approximately 150,000 Da. According to SDS-PAGE WGeIF-2 consists of four subunits with M(r) 37,000 (alpha), 40,000 (beta), 42,000 (gamma) and 52,000 (delta). The beta- and gamma-subunits (but not the alpha-subunit) of WGeIF-2 can be readily phosphorylated by the double-stranded RNA activated kinase isolated from rabbit reticulocytes. Dissociation constants for WGeIF-2 complexes with GDP and GTP were measured. In our evaluation the WGeIF-2 affinity for GDP (KdGDP = 1.5 x 10(-7) M) was only 10 times higher than for GTP (KdGTP = 1.5 x 10(-6) M), while for rabbit reticulocyte eIF-2 (RReIF-2) the difference has been estimated as as much as two orders of magnitude in accordance with the literature. Close values of dissociation constants for WGeIF-2 complexes with guanine nucleotides suggest that at a sufficiently high [GTP]/[GDP] ratio the nucleotide exchange in wheat cells may take place without the participation of specific factor (eIF-2B) which catalyzes the nucleotide exchange on eIF-2 from mammalian cells.     

The alpha and gamma subunits of initiation factor eIF-2 can be cross-linked to 18S ribosomal RNA within the quaternary initiation complex, eIF-2.Met-tRNAf.GDPCP.small ribosomal subunit.

Initiation factor eIF-2 from rat liver was reacted with the hetero-bifunctional cross-linking reagents ABAI or APTPI without diminishing its ability to form the quaternary initiation complex with Met-tRNAf, GDPCP and the small ribosomal subunit. Upon irradiation with UV light, subunits alpha and gamma of eIF-2 became covalently linked to 18S ribosomal RNA. The subunits were identified electrophoretically after isolation of the covalent protein-rRNA complexes and subsequent degradation of the rRNA by nuclease and alkali treatments. The close proximity of the two factor subunits to sequences of ribosomal RNA within the quaternary complex could be confirmed in a second set of experiments using unmodified, 125I-labeled factor and diepoxybutane as cross-linking reagent.     

Ligand interactions with eukaryotic translation initiation factor 2: role of the gamma-subunit.

Eukaryotic translation initiation factor 2 (eIF-2) comprises three non-identical subunits alpha, beta and gamma. In vitro, eIF-2 binds the initiator methionyl-tRNA in a GTP-dependent fashion. Based on similarities between eukaryotic eIF-2gamma proteins and eubacterial EF-Tu proteins, we previously proposed a major role for the gamma-subunit in binding guanine nucleotide and tRNA. We have tested this hypothesis by examining the biochemical activities of yeast eIF-2 purified from wild-type strains and strains harboring mutations in the eIF-2gamma structural gene (GCD11) predicted to alter ligand binding by eIF-2. The alteration of tyrosine 142 in yeast eIF-2gamma, corresponding to histidine 66 in Escherichia coli EF-Tu, dramatically reduced the affinity of eIF-2 for Met-tRNAi(Met) without affecting the k(off) value for guanine nucleotides. In contrast, non-lethal substitutions at a conserved lysine residue (K250) in the putative guanine ring-binding loop increased the off-rate for GDP, thereby mimicking the function of the guanine nucleotide exchange factor eIF-2B, without altering the apparent dissociation constant for Met-tRNAi(Met). For eIF-2[gamma-K250R], the increased off-rate also seen for GTP was masked by the presence of Met-tRNAi(Met) in vitro. In vivo, increasing the dose of the yeast initiator tRNA gene suppressed the slow-growth phenotype and reduced GCN4 expression in gcd11-K250R and gcd11-Y142H strains. These studies indicate that the gamma-subunit of eIF-2 does indeed provide EF-Tu-like function to the eIF-2 complex, and further suggest that the level of Met-tRNAi(Met) is critical for maintaining wild-type rates of initiation in vivo.     

A novel role for aminoacyl-tRNA synthetases in the regulation of polypeptide chain initiation

Exposure of the temperature-sensitive leucyl-tRNA synthetase mutant of Chinese hamster ovary cells, tsH1, to the non-permissive temperature of 39.5 degrees C results in a rapid inhibition of polypeptide chain initiation. This inhibition is caused by a reduced ability of the eukaryotic initiation factor eIF-2 to participate in the formation of eIF-2.GTP.Met-tRNAf ternary complexes and thus in the formation of 43S ribosomal pre-initiation complexes. Associated with this decreased eIF-2 activity is an increased phosphorylation of the eIF-2 alpha subunit. It has previously been shown in other systems that phosphorylation of eIF-2 alpha slows the rate of recycling of eIF-2.GDP to eIF-2.GTP catalysed by the guanine nucleotide exchange factor eIF-2B. We show here that phosphorylation of eIF-2 alpha by the reticulocyte haem-controlled repressor also inhibits eIF-2B activity in cell-free extracts derived from tsH1 cells. Thus the observed increased phosphorylation of eIF-2 alpha at the non-permissive temperature in this system is consistent with impaired recycling of eIF-2 in vivo. Using a single-step temperature revertant of tsH1 cells, TR-3 (which has normal leucyl-tRNA synthetase activity at 39.5 degrees C), we demonstrate here that all inhibition of eIF-2 function reverts together with the synthetase mutation. This establishes the close link between synthetase function and eIF-2 activity. In contrast, recharging tRNALeu in vivo in tsH1 cells at 39.5 degrees C by treatment with a low concentration of cycloheximide failed to reverse the inhibition of eIF-2 function. This indicates that tRNA charging per se is not involved in the regulatory mechanism. Our data indicate a novel role for aminoacyl-tRNA synthetases in the regulation of eIF-2 function mediated through phosphorylation of the alpha subunit of this factor. However, in spite of the fact that cell-free extracts from Chinese hamster ovary cells contain protein kinase and phosphatase activities active against either exogenous or endogenous eIF-2 alpha, we have been unable to show any activation of kinase or inactivation of phosphatase following incubation of the cells at 39.5 degrees C.     

Mutations in GCD11, the structural gene for eIF-2 gamma in yeast, alter translational regulation of GCN4 and the selection of the start site for protein synthesis.

Translation initiation factor 2 (eIF-2) in eukaryotic organisms is composed of three non-identical subunits, alpha, beta and gamma. In a previous report, we identified GCD11 as an essential gene encoding the gamma subunit of eIF-2 in the yeast Saccharomyces cerevisiae. The predicted amino acid sequence of yeast eIF-2 gamma displays remarkable similarity to bacterial elongation factor Tu, including the presence of sequence elements conserved in all known guanine nucleotide binding proteins. We have identified the molecular defects present in seven unique alleles of GCD11 characterized by a partial loss of function. Three of these mutations result in amino acid substitutions within the putative GTP binding domain of eIF-2 gamma. We show that the gcd11 mutations specifically alter regulation of GCN4 expression at the translational level, without altering the scanning mechanism for protein synthesis initiation. Six of the mutant alleles presumably alter the function of eIF-2 gamma, rather than its abundance. A single allele, gcd11-R510H, suppresses a mutant his4 allele that lacks a functional AUG start codon. The latter result indicates that the gamma subunit of eIF-2 participates in recognition of the start site for protein synthesis, a role previously demonstrated in yeast for eIF-2 alpha and eIF-2 beta.     

Effect of starvation and diabetes on the activity of the eukaryotic initiation factor eIF-2 in rat skeletal muscle

The ability of the initiation factor eIF-2 in skeletal muscle extracts to form ternary initiation complexes ([Met-tRNA(f).eIF-2.GDP]) is decreased by either starvation or diabetes. These conditions also impair the ability of muscle extracts to dissociate [eIF-2.GDP], suggesting inhibition of the guanine nucleotide exchange reaction essential for eIF-2 recycling. We could not, however, detect any change in the phosphorylation state of the alpha subunit of eIF-2. This suggests that eIF-2 activity may be regulated in this system by a mechanism not involving its phosphorylation.     

Phosphorylation of initiation factor eIF-2 alpha, binding of mRNA to 48 S complexes, and its reutilization in initiation of protein synthesis

The formation of 80 S initiation complexes containing labeled viral mRNA was drastically inhibited when mRNA binding assays were carried out with reticulocyte lysate preincubated with double-stranded RNA (dsRNA). When the assays were analyzed by centrifugation on sucrose gradients, the mRNA incubated with lysate pretreated with dsRNA sedimented as a 48 S complex. Met-tRNA, GDP, and phosphorylated initiation factor eIF-2(alpha P) were shown to co-sediment with the 48 S complex. Therefore, the formation of this complex was attributed to the phosphorylation of eIF-2 alpha by a dsRNA-activated protein kinase. These observations suggested that mRNA could bind to a 40 S ribosomal subunit containing Met-tRNAf, GDP, and eIF-2(alpha P), but the joining of a 60 S ribosomal subunit was inhibited. When the 48 S complex was isolated and incubated with lysate without added dsRNA, the mRNA could form 80 S initiation complexes. The shift of mRNA from 48 S to 80 S complexes was also observed when the eIF-2 alpha kinase activity was inhibited by the addition of 2-aminopurine. This shift was quite slow, however, when compared to the rate of binding of free mRNA to 80 S initiation complexes. The 2-aminopurine was effective in reversing the inhibition of protein synthesis by dsRNA and in maintaining a linear rate of protein synthesis for 3 h in lysates. Without added 2-aminopurine, protein synthesis was inhibited after 90 min even in lysates supplemented with hemin and eIF-2(alpha P) was detected in these lysates. This finding indicated that eIF-2 alpha phosphorylation could be in part responsible for limiting the duration of protein synthesis in mammalian cell-free systems.     

Synthesis of human initiation factor-2 alpha in Saccharomyces cerevisiae.

A human eIF-2 alpha cDNA (encoding alpha-subunit of the eukaryotic initiation factor-2) was expressed under the control of the galactose-regulated GAL1, 10 promoter, in Saccharomyces cerevisiae, in order to study the possible interactions of human eIF-2 alpha with the yeast protein synthesis apparatus. Isoelectric focusing coupled with Western-blot analysis demonstrated that the human eIF-2 alpha subunit synthesized in yeast under a variety of growth conditions was detected as two bands which co-migrated with the phosphorylated and unphosphorylated forms of rabbit eIF-2 alpha, suggesting covalent modification in vivo. Cell fractionation studies further demonstrated that the synthesised human eIF-2 alpha protein, though present in the cytoplasm, was largely associated with the yeast ribosomes, but could be removed from these by washing with 0.3 M KCl. This possible association of the synthesised human subunit into a three-subunit (alpha, beta and gamma) eIF-2 complex was further examined by partial purification of the yeast eIF-2 complex and estimation of the molecular mass of this complex. Immunoreactive eIF-2 alpha was found in fractions with eIF-2 activity and the estimated molecular mass (130 kDa) corresponded to that predicted for the eIF-2 trimer. These analyses suggest that human eIF-2 alpha subunit synthesised in yeast can become involved with the yeast protein synthetic apparatus, though whether this is a functional incorporation requires further genetic studies.     

Stimulation of protein synthesis in COS cells transfected with variants of the alpha-subunit of initiation factor eIF-2.

The role of eukaryotic initiation factor 2 (eIF-2) phosphorylation in translational control has been demonstrated in vivo by overexpressing variant forms of eIF-2 alpha that are not phosphorylated. COS-1 cells transiently transfected with expression vectors for human eIF-2 alpha contain 10-20-fold more eIF-2 alpha subunit than the endogenous COS cell eIF-2 trimeric complex. Expression of the variant form of eIF-2 alpha, Ser51Asp, where Asp replaces Ser51, causes inhibition of protein synthesis, whereas the Ser48Asp variant does not. When either Ser48 or Ser51 is replaced by Ala, the variants stimulate dihydrofolate reductase synthesis when the eIF-2 alpha kinase, DAI, is activated. In order to elucidate these mechanisms, we have separated eIF-2 trimeric complexes from free overexpressed eIF-2 alpha subunits by fast protein liquid chromatography Superose chromatography. Pulse-labeled cells transfected with wild-type or variant DNAs produced eIF-2 preparations with greater than 10-fold higher specific radioactivity in the alpha-subunit compared to the gamma-subunit, thus demonstrating that the human eIF-2 alpha produced from the plasmids readily exchanges into COS cell eIF-2 complexes. Both wild-type and Ser48Ala variant forms of the free 2 alpha-subunit, further purified by MonoQ chromatography, are poor substrates for the heme-regulated eIF-2 alpha kinase, HRI, but are good substrates for double-stranded RNA-activated inhibitor in vitro; the Ser51Ala variant subunit is not phosphorylated by either kinase. None of the purified free eIF-2 alpha subunits inhibits phosphorylation of eIF-2 in vitro, even at up to 8-fold molar excess. Examination of the extent of eIF-2 alpha phosphorylation in the COS cell eIF-2 complexes by two-dimensional polyacrylamide gel electrophoresis shows that the stimulation of dihydrofolate reductase synthesis by the Ser51Ala variant is most readily explained by failure of eIF-2 to be phosphorylated. Stimulation by the Ser48Ala variant appears to occur by mitigation of the effect of phosphorylation at Ser51 since the double variant, Ser48Ala-Ser51Asp, inhibits protein synthesis less than the single variant Ser51Asp. The evidence argues strongly against there being a second site of phosphorylation involved in translational repression.     

Partial purification and characterization of reticulocyte phosphatase with activity for phosphorylated peptide initiation factor 2

An enzyme fraction containing phosphatase activity for phosphorylated eukaryotic peptide initiation factor 2 (eIF-2) has been isolated from rabbit reticulocytes and partially characterized. The enzyme efficiently catalyzes release of phosphate from the small subunit of eIF-2 (eIF-2 alpha) that has been phosphorylated by the hemin-controlled repressor. It is shown to restore activity of this phosphorylated eIF-2 for binding of methionyl-tRNAf to 40 S ribosomal subunits in a partial reaction of peptide initiation. The enzyme fraction also has phosphatase activity for eIF-2 phosphorylated in its largest subunit and for the 100,000-dalton peptide associated with the eIF-2 alpha kinase activity of the hemin-controlled repressor. The phosphoprotein phosphatase has been isolated by a procedure involving precipitation with ethanol at room temperature and has an apparent molecular weight in the order of 76,000. Its phosphatase activity for eIF-2 alpha is stimulated about 3-fold by optimal concentrations of Mn2+, but is not stimulated by Ca2+ or Mg2+. The enzyme is strongly inhibited by Fe2+ and by purine nucleoside diphosphates.     

Binding and release of radiolabeled eukaryotic initiation factors 2 and 3 during 80 S initiation complex formation.

The AUG-dependent formation of an 80 S ribosomal initiation complex was studied using purified rabbit reticulocyte initiation factors radiolabeled by reductive methylation. The radiolabeled initiation factors were as biologically active as untreated factors. Reaction mixtures containing a variety of components (AUG, GTP, Met-tRNAf, initiation factors, and 40 S and 60 S ribosomal subunits) were incubated at 30 degrees C and then analyzed on linear sucrose gradients for the formation of ribosomal complexes. The results show that both eukaryotic initiation factor (eIF)-3 and the ternary complex (eIF-2.GTP.Met-tRNAf) bind independently to the 40 S subunit and each of these components enhances the binding of the other. All of the polypeptides of eIF-2 and eIF-3 participate in this binding. Formation of an 80 S ribosomal complex requires eIF-5 and 60 S subunits in a reaction that is stimulated by eIF-4C. Both eIF-2 and eIF-3 are released from the 40 S preinitiation complex during formation of the 80 S initiation complex. Release of eIF-2 and eIF-3 does not occur and 80 S ribosomal complexes are not formed if GTP is replaced by a nonhydrolyzable analog such as guanosine 5'-O3-(1,2-mu-imido)triphosphate. Despite a variety of attempts, it has not yet been possible to demonstrate binding of eIF-4C, eIF-4D, or eIF-5 to either 40 S or 80 S ribosomal complexes.